The introduction of electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) techniques helped mass spectrometry to experience a giant leap in the analytical chemistry of large molecules and biopolymers during the last two decades. Mass spectrometers with higher-accuracy mass detection capability (such as Fourier transform ion cyclotron resonance mass spectrometers, FT-ICR MS, or electrospray orthogonal time-of-flight mass spectrometers ESI-TOF MS) satisfied some needs for the accurate mass information. Additionally, different MS/MS capabilities allowed to obtain important structural information upon fragmentation of selected ions especially by collisions with neutral particles (collision induced dissociation, CID), by sequential absorption of multiple infrared photons from a CO2 laser (infrared multiphoton dissociation, IRMPD), by capturing low energy electrons (electron capture dissociation, ECD) or by electron transfer from a negative ion (electron transfer dissociation, ETD).
In biochemistry, particularly for characterization of proteins, the amino acid sequence is important analytical information. However, the physiologic activity of a protein does not only depend on its amino acid sequence. Protein chains fold to form secondary structures (alpha helices, beta sheets), which in turn fold further and build tertiary structures in order to take more stable conformations. Additionally, in some cases, a number of folded protein units form non-covalent agglomerates (quaternary structures): Hemoglobin for example forms its characteristic tetrameric structure with two alpha and two beta chains.
Recent experience with proteins teaches that neurodegenerative disorders like Alzheimer's, Parkinson's, Huntington's diseases, Amyotrophic lateral sclerosis (ALS), and transmissible spongiform encephalopathies (TSE) like bovine spongiform encephalopathy (BSE) or the human equivalent Creutzfeld-Jakob disease (CJD) are closely related to misfolding of proteins. Thus, studies to obtain information about tertiary protein structures became crucial. For discovering physiologic activities of proteins, the information about their folding geometry is complementary to their basic amino acid sequence information.
Overall geometric changes of proteins are also observed due to other effects: Many proteins are post translationally modified. About 70% of the active proteins in human organism are glycosylated. Posttranslational modifications (PTMs) also induce differences in protein geometry. Modified proteins containing a number of polar groups like phosphates or sugar groups, both steric and electronic interactions within the molecule increase its potential energy. The molecule undergoes conformational changes to accommodate the new groups without losing much of its stability. Therefore, phosphorylated and glycosylated proteins can have different overall geometry than their unmodified analogues. PTMs obviously alter the mass of the protein, and thus, the overall mass of a modified protein will be different. Proteins that have just some small differences, e.g. some post translational modifications, are isoforms. Isoforms can also have very different geometric cross sections. Structural characterization of various isoforms of large proteins, in terms of determining positions, number, and kind of particular modifications can be difficult using the mostly applied fragmentation (MS/MS) methods (CID, IRMPD), since during these processes the PTM groups are not protected. Only ECD and ETD methods can protect the PTMs, but both of them are limited to certain types of mass spectrometers.
Structural or conformational isomers have different geometric appearance but exactly the same mass. Thus, they cannot be recognized as different species in regular mass spectrometry. One of the most efficient ways to recognize and separate structural or conformational isomers is the ion mobility separation. Ions are accelerated in an ion mobility cell. An ion mobility cell has an inert collision gas (for example helium). Ions are accelerated in an electric field. Due to the collisions with gas molecules the ions are also exposed to a drag force and therefore move through the cell with a constant velocity proportional to the electric field. The proportionality constant is called “ion mobility” and is a function of the temperature, pressure, ion charge, ion collision cross section, and the reduced mass. Ions with the same mass but with different collisions cross sections have different mobilities. If different conformational isomers of the same compound are accelerated in an ion mobility cell, the isomer with the smallest geometric cross section will have the highest ion mobility. Protonated molecules of a tightly folded protein conformer have smaller geometric cross section and therefore will be exposed during their flight through the mobility cell a smaller number of collisions. Ions with open (unfolded) conformation will be exposed to a larger number of collisions and therefore fly slower than the tightly folded isomer with the same mass to charge ratio. Unfolded isomers exit the mobility cell at a later “arrival time” (as illustrated in FIGS. 1a and 1b, described below).
The information to be extracted from ion mobility separation measurements is in various levels. Ions of exactly the same mass-to-charge ratio but with different conformation will be separated. From absolute values of the ion mobility cross sections in a certain collision gas (e.g. helium) various ion conformation possibilities can be calculated using available force field programs e.g. AMBER (Assisted Model Building and Energy Refinement) or CHARMM (Chemistry at HARvard Macromolecular Mechanics). Measurement of relative geometric cross sections is also very often applied in the ion mobility separated mass spectrometry for studying changes and for discovering the existence of different isomeric structures.
In the past, the main application of ion mobility spectrometers was the detection of chemical warfare agents, drugs, and explosives. Ion mobility spectrometers specifically built for on site detections of these compound classes are usually small hand-held detectors. The mobility-separation process of ions in these devices is performed in a collision gas. Unlike for mass spectrometers, no vacuum is required in these ion mobility spectrometers. Thus, no expensive pumping systems are needed in these devices, and the production of them is relatively inexpensive.
In the recent years, as the importance of the ion mobility separation has increased for chemical and biological research, ion mobility separation cells were integrated to mass spectrometric systems to combine the ionic cross section information of isomeric compounds with their mass information. One major drawback of these hybrid instruments is the high pressure range of the ion mobility separation part, which is not really compatible with the rest of the mass spectrometric equipment. For decent ion mobility separations, pressures of 1 mbar or higher are required, where mostly helium is used as a collision gas (sometimes argon is also used). High pressure cells in mass spectrometric vacuum systems have always been a challenge for pumping. Large pumping units are crucial to pump out the collision gas in the high pressure chamber. Additionally, high pressure chambers have to be isolated from the rest of the mass spectrometer by carefully designed pumping stages. These measures increase the required space to accommodate the ion mobility separators, complicate the construction these hybrid systems and the cost of the final equipment. Solutions always involve compromises on both sides.
Further problems arise from coupling the ion mobility separation cell to a succeeding analyzer (e.g. mass analyzers) concerning the timing of the mobility-separated ions leaving the ion mobility separation cell and entering the analyzer (e.g. mass analyzer). The succeeding analyzer must generally be much faster than the ion mobility separation which is even not easily achievable for fast scanning mass analyzers like time-of-flight mass spectrometers with orthogonal ion injection (OTOF-MS). If the mobility cell has conventional dimensions, the time separation of, for instance, the conformational isomers of proteins are in the range of hundreds of microseconds.
As mentioned, this separation time becomes, even for the time-of-flight mass spectrometers, a challenging problem. Other mass spectrometers are much slower. In the case of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), the time from the ionization process to the detection time can be several hundred milliseconds to several seconds. There is practically no way to fully use ion mobility separation spectrum. The only way to use an ion mobility-FT-ICR MS hybrid instrument is to apply a so-called slicing method. In this method ions of a predefined ion mobility window can be trapped and collected in a linear RF multipole ion trap and then are transferred into the ion cyclotron resonance cell for analysis. An on-line analysis of a complete ion mobility spectrum is not possible here due to time reasons.
In regular ion mobility spectrometers, the ion mobility separation cell has an ion detection device at the exit. In recent years ion mobility separation has been combined with mass spectrometry so that the mobility separated isomers can be mass analyzed by mass spectrometry. U.S. Pat. No. 5,905,258 (David E. Clemmer and James P. Reilly) and U.S. Pat. No. 6,960,761 (David E. Clemmer) describe mass spectrometric systems equipped with ion mobility separation devices.